Rapamycins are novel anticancer agents that specifically inhibit the activity of a protein Ser/Thr kinase (mTOR) that controls translation of proteins involved in cell cycle progression and survival. At present two analogues, CCI-779 and RAD001, are in clinical development, and have shown antitumor activity in phase I and II trials. In the last period of funding our studies demonstrated that rapamycin induces apoptosis only in cells lacking a functional p53/p21Cip1 pathway. This, therefore, presents a biochemical rationale for tumor-selective killing. Rapamycin-induced apoptosis is a consequence of mTOR inhibition that rapidly induces cellular stress, characterized by activation of apoptosis signaling kinase 1 (ASK1). This leads to activation of Jun N-terminal kinase, and increased levels of phospho-c-Jun. Of importance is the observation that this stress response is rapidly suppressed in cells with functional p53 in a p21Cip1 dependent manner. However, suppression of apoptosis is not a consequence of p53/p21Cip1mediated cell cycle arrest. Rather, rapamycin treatment leads to the association of ASK1 with p21Cip1 and suppression of ASK1 activity. Preliminary results demonstrate that amino acid deprivation mimics the rapamycin-induced stress response. In this application we propose a series of studies that will determine: 1. Whether the rapamycin-induced stress response is a consequence of inhibition of mTOR kinase function, and results from inhibition of signaling to 4E-BP, S6K1 or protein phosphatase 5 (PP5). 2. How rapamycin treatment induces binding of p21Cip1 to ASK1, and determine whether activation of Jun N-terminal kinase (JNK) or elevated P-c-Jun is required for complex formation. 3. Domains of p21Cip1 and ASK1 required for complex formation. 4. Whether the cellular stress induced by amino acid deprivation is similar to that induced by rapamycin, and determine whether the p53/p21 axis is required to suppress nutritional stress. We propose that the rapamycin-induced stress response is a fundamental response to mTOR inhibition, and that p53/p21Cip1 function is essential to protect cells from apoptosis in nutritionally deficient environments. The work proposed in this application will help elucidate the synthetic lethal interaction that is induced by rapamycins only in cells with mutations that predispose to cancer formation, i.e. p53 or p21Cip1. Our studies will develop the biochemical framework to underpin a novel approach by which rapamycins can induce tumor-selective cytotoxicity based on the loss of the tumor suppressor p53.